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Abstract Background Primary sheep fetal fibroblasts (SFFCs) have emerged as a valuable resource for investigating the molecular and pathogenic mechanisms of orf viruses (ORFV). However, their utilization is considerably restricted due to the exorbitant expenses associated with their isolation and culture, their abbreviated lifespan, and the laborious procedure. Results In our investigation, the primary SFFCs were obtained and immortalized by introducing a lentiviral recombinant plasmid containing the large T antigen from simian virus 40 (SV40). The expression of fibronectin and vimentin proteins, activity of SV40 large T antigen, cell proliferation assays, and analysis of programmed cell death revealed that the immortalized large T antigen SFFCs (TSFFCs) maintained the same physiological characteristics and biological functions as the primary SFFCs. Moreover, TSFFCs demonstrated robust resistance to apoptosis, extended lifespan, and enhanced proliferative activity compared to primary SFFCs. Notably, the primary SFFCs did not undergo in vitro transformation or exhibit any indications of malignancy in nude mice. Furthermore, the immortalized TSFFCs displayed live ORFV vaccine susceptibility. Conclusions Immortalized TSFFCs present valuable in vitro models for exploring the characteristics of ORFV using various techniques. This indicates their potential for secure utilization in future studies involving virus isolation, vaccine development, and drug screening.

The Egyptian Rousettus bat (Rousettus aegyptiacus) is a common fruit bat species that is distributed mainly in Africa and the Middle East. Bats serve as reservoir hosts for numerous pathogens. Human activities, such as hunting bats for food, managing vermin, and causing habitat loss, elevate the likelihood of transmission of bat pathogens to humans and other animals. Consequently, bat cell lines play a crucial role as research materials for investigating viral pathogens. However, the inherent limitation of finite cell division in primary cells necessitates the use of immortalized cells derived from various bat tissues. Herein, we successfully established six fibroblast cell lines derived from an infant bat heart and lungs and an elderly bat heart. Three of the six cell lines, called K4DT cells, were transduced by a combination of cell cycle regulators, mutant cyclin‐dependent kinase 4, cyclin D1, and human telomerase reverse transcriptase. The other three cell lines, named SV40 cells, were transfected with simian virus 40 large T antigen. Transgene protein expression was detected in the transduced cells. All three K4DT cell lines and one lung‐derived SV40 cell line were virtually immortalized and nearly maintained the normal diploid karyotypes. However, the two other heart‐derived SV40 cell lines had aberrant karyotypes and the young bat‐derived cell line stopped proliferating at approximately 40 population doublings. These bat cell lines are valuable for studying pathogen genomics and biology.

The Egyptian Rousettus bat (Rousettus aegyptiacus) is a common fruit bat species that is distributed mainly in Africa and the Middle East. Bats serve as reservoir hosts for numerous pathogens. Human activities, such as hunting bats for food, managing vermin, and causing habitat loss, elevate the likelihood of transmission of bat pathogens to humans and other animals. Consequently, bat cell lines play a crucial role as research materials for investigating viral pathogens. However, the inherent limitation of finite cell division in primary cells necessitates the use of immortalized cells derived from various bat tissues. Herein, we successfully established six fibroblast cell lines derived from an infant bat heart and lungs and an elderly bat heart. Three of the six cell lines, called K4DT cells, were transduced by a combination of cell cycle regulators, mutant cyclin‐dependent kinase 4, cyclin D1, and human telomerase reverse transcriptase. The other three cell lines, named SV40 cells, were transfected with simian virus 40 large T antigen. Transgene protein expression was detected in the transduced cells. All three K4DT cell lines and one lung‐derived SV40 cell line were virtually immortalized and nearly maintained the normal diploid karyotypes. However, the two other heart‐derived SV40 cell lines had aberrant karyotypes and the young bat‐derived cell line stopped proliferating at approximately 40 population doublings. These bat cell lines are valuable for studying pathogen genomics and biology. [ABSTRACT FROM AUTHOR]

Cellular eukaryotic replication initiation helicases are first loaded as head-to- head double hexamers on double-stranded (ds) DNA origins and then initiate S-phase DNA melting during licensed (once per cell cycle) replication. Merkel cell polyomavirus (MCV) large T (LT) helicase oncoprotein similarly binds and melts its own 98-bp origin but replicates multiple times in a single cell cycle. To examine the actions of this unlicensed viral helicase, we quantitated multimerization of MCV LT molecules as they assembled on MCV DNA origins using real-time single-molecule microscopy. MCV LT formed highly stable double hexamers having 17-fold longer mean lifetime (τ, >1,500 s) on DNA than single hexamers. Unexpectedly, partial MCV LT assembly without double-hexamer formation was sufficient to melt origin dsDNA as measured by RAD51, RPA70, or S1 nuclease cobinding. DNA melting also occurred with truncated MCV LT proteins lacking the helicase domain, but was lost from a protein without the multimerization domain that could bind only as a monomer to DNA. SV40 polyomavirus LT also multimerized to the MCV origin without forming a functional hexamer but still melted origin DNA. MCV origin melting did not require ATP hydrolysis and occurred for both MCV and SV40 LT proteins using the nonhydrolyzable ATP analog, adenylyl-imidodiphosphate (AMP-PNP). LT double hexamers formed in AMP-PNP, and melted DNA, consistent with direct LT hexamer assembly around single-stranded (ss) DNA without the energy-dependent dsDNA-to- ssDNA melting and remodeling steps used by cellular helicases. These results indicate that LT multimerization rather than helicase activity is required for origin DNA melting during unlicensed virus replication. [ABSTRACT FROM AUTHOR]

Skin injury is repaired through a multi-phase wound healing process of tissue granulation and re-epithelialization. Any failure in the healing process may lead to chronic non-healing wounds or abnormal scar formation. Although significant progress has been made in developing novel scaffolds and/or cell-based therapeutic strategies to promote wound healing, effective management of large chronic skin wounds remains a clinical challenge. Keratinocytes are critical to re-epithelialization and wound healing. Here, we investigated whether exogenous keratinocytes, in combination with a citrate-based scaffold, enhanced skin wound healing. We first established reversibly immortalized mouse keratinocytes (iKera), and confirmed that the iKera cells expressed keratinocyte markers, and were responsive to UVB treatment, and were non-tumorigenic. In a proof-of-principle experiment, we demonstrated that iKera cells embedded in citrate-based scaffold PPCN provided more effective re-epithelialization and cutaneous wound healing than that of either PPCN or iKera cells alone, in a mouse skin wound model. Thus, these results demonstrate that iKera cells may serve as a valuable skin epithelial source when, combining with appropriate biocompatible scaffolds, to investigate cutaneous wound healing and skin regeneration.

The initiation of Okazaki fragment synthesis during cellular DNA replication is a crucial step for lagging strand synthesis, which is carried out by the primase function of DNA polymerase α‐primase (Pol‐prim). Since cellular replication protein A (RPA) prevents primase from starting RNA synthesis on single‐stranded DNA (ssDNA), primase requires auxiliary factors, such as the simian virus 40 (SV40) T antigen (Tag), for the initiation reaction on RPA‐bound ssDNA. Here, we investigated the ability of Tag variants and Tag protein complexes to bind to ssDNA and their resulting effects on the stimulation of Pol‐prim on free and RPA‐bound ssDNA. Atomic force microscopy imaging showed that while Tag131‐627(V350E/P417D) and Tag131‐627(L286D/R567E) (abbreviated as M1 and M2, respectively) could bind to ssDNA as monomers, these monomeric Tags could come together and bind to ssDNA as dimers as well. In a model assay for the initiation of Okazaki fragment synthesis, full‐length Tag SV40 Tag1‐708 and monomeric M2 stimulated DNA synthesis of Pol‐prim on ssDNA and on RPA‐bound ssDNA. In contrast, neither monomeric M1 nor M1‐M2 dimers could stimulate Pol‐prim, on ssDNA or on RPA‐bound ssDNA. Overall, we show that a lack of stimulatory activity of monomeric M1 and M1‐M2 dimers suggests that residues V350 and P417 are not only important for interactions between Tag molecules but also for protein–protein interactions within Okazaki fragment initiation complexes. Thus, we highlight that mutations in M1 are dominant negative with regard to Okazaki fragment initiation.

Although uncoupling protein 1 (UCP1) is established as a major contributor to adipose thermogenesis, recent data have illustrated an important role for alternative pathways, particularly the futile creatine cycle (FCC). How these pathways co-exist in cells and tissues has not been explored. Beige cell adipogenesis occurs in vivo but has been difficult to model in vitro ; here, we describe the development of a murine beige cell line that executes a robust respiratory response, including uncoupled respiration and the FCC. The key FCC enzyme, tissue-nonspecific alkaline phosphatase (TNAP), is localized almost exclusively to mitochondria in these cells. Surprisingly, single-cell cloning from this cell line shows that cells with the highest levels of UCP1 express little TNAP, and cells with the highest expression of TNAP express little UCP1. Immunofluorescence analysis of subcutaneous fat from cold-exposed mice confirms that the highest levels of these critical thermogenic components are expressed in distinct fat cell populations. [Display omitted] • Development of a beige fat cell line containing both UCP1 and the futile creatine cycle • Clonal analysis reveals different subclasses of beige adipocytes • Identification of UCP1high and TNAPhigh adipocytes in different fat cells in vivo • Detection of TNAPhigh adipocyte population in human deep neck brown fat Different thermogenic mechanisms have been described; how they relate to each other is unknown. Vargas-Castillo et al. developed a beige fat cell line containing both UCP1 and the futile creatine cycle. Clonal and in vivo analyses revealed the highest levels of these critical thermogenic components are expressed in distinct fat cell populations. [ABSTRACT FROM AUTHOR]

Background: Heat shock response is a protected mechanism against environmental changes for the organism, which must be tightly regulated. Bromodomain and extra terminal-containing protein family (BETs) regulate numerous gene expression in many physiological and pathological conditions, including viral infection. SV40 is considered as a highly human disease-associated virus. Objective: We aimed to explore whether BETs play a role in heat shock in SV40 large T antigen transfected cells. Methods: SV40LTA was transfected in HeLa cells using the Lipofectamine 8000. BETs inhibitor JQ1 and I-BET-762 was employed to treat transfected cells and HEK-293 T cells. Heat shock treatment was performed to determine the effect of JQ1 and I-BET-762 on these cells. Western blot and quantitative RT-PCR were carried out to assess the expression of HSP70 and other HSPs. Results: We found that inhibition of BETs by JQ1 and I-BET-762 protects cells from heat shock-induced death in HEK293T cells. Both JQ1 and I-BET-762 induce the expression of HSPs and HSF1 in HEK-293 T cells. However, neither JQ1 nor I-BET-762 fail to induce the expression of HSPs in either HeLa or HBL-1 cells. When SV40 large T antigen was transfected into HeLa cells, the induction of HSP70 expressing and the protection of heat shock-induced cell death are reproduced by JQ1 and IBET treatment in these transfected cells. Conclusions: Inhibition of BETs by JQ1 and I-BET-762 prevents heat shock-induced cell death via upregulating HSPs in SV40 large T antigen transfected cells. Our data indicate a novel function of BETs in SV40 large T antigen transformed cells, affecting HSPs and HSF1 as well as its function on heat shock response. [ABSTRACT FROM AUTHOR]

BACKGROUND: Cell immortalization is a characteristic of cells acquiring the ability to continue to proliferate. The large T antigen gene can improve the efficiency of cell immortalization, and is widely used in experiments, attracting more and more attention from researchers. OBJECTIVE: To establish an immortalized cell line of human bone marrow mesenchymal stem cells and lay the foundation for the clinical application of human bone marrow mesenchymal stem cells. METHODS: The recombinant plasmid vector containing the SV40 large T antigen gene was used to transfect human bone marrow mesenchymal stem cells, which were successively subcultured and identified by methods, such as morphology, cell proliferation ability, cell surface markers, and multidirectional differentiation potential. RESULTS AND CONCLUSION: (1) The immortalized cell line constructed by this method is an adherent growth of spindle cells with specific surface markers of human bone marrow mesenchymal stem cells. (2) The 50th generation of human bone marrow mesenchymal stem cells can still express SV40 large T antigen gene after transfection. (3) The proliferation ability of the immortalized cell line is better than that of untransfected human bone marrow mesenchymal stem cells. (4) The immortalized cell line has the potential to differentiate into osteogenic, adipogenic, and chondrogenic cells. (5) This shows that transfection of the SV40 large T antigen gene does not affect the biological characteristics and differentiation ability of human bone marrow mesenchymal stem cells. This method can be used to establish immortalized human bone marrow mesenchymal stem cells. [ABSTRACT FROM AUTHOR]

The initiation of Okazaki fragment synthesis during cellular DNA replication is a crucial step for lagging strand synthesis, which is carried out by the primase function of DNA polymerase α‐primase (Pol‐prim). Since cellular replication protein A (RPA) prevents primase from starting RNA synthesis on single‐stranded DNA (ssDNA), primase requires auxiliary factors, such as the simian virus 40 (SV40) T antigen (Tag), for the initiation reaction on RPA‐bound ssDNA. Here, we investigated the ability of Tag variants and Tag protein complexes to bind to ssDNA and their resulting effects on the stimulation of Pol‐prim on free and RPA‐bound ssDNA. Atomic force microscopy imaging showed that while Tag131‐627(V350E/P417D) and Tag131‐627(L286D/R567E) (abbreviated as M1 and M2, respectively) could bind to ssDNA as monomers, these monomeric Tags could come together and bind to ssDNA as dimers as well. In a model assay for the initiation of Okazaki fragment synthesis, full‐length Tag SV40 Tag1‐708 and monomeric M2 stimulated DNA synthesis of Pol‐prim on ssDNA and on RPA‐bound ssDNA. In contrast, neither monomeric M1 nor M1‐M2 dimers could stimulate Pol‐prim, on ssDNA or on RPA‐bound ssDNA. Overall, we show that a lack of stimulatory activity of monomeric M1 and M1‐M2 dimers suggests that residues V350 and P417 are not only important for interactions between Tag molecules but also for protein–protein interactions within Okazaki fragment initiation complexes. Thus, we highlight that mutations in M1 are dominant negative with regard to Okazaki fragment initiation. [ABSTRACT FROM AUTHOR]

Abstract Background Spermatogonial stem cells (SSCs) are capable of both self-renewal and differentiation to mature functional spermatozoa, being the only adult stem cells in the males that can transmit genetic information to the next generation. Porcine SSCs hold great value in transgenic pig production and in establishment of porcine models for regenerative medicine. However, studies and applications of porcine SSCs have been greatly hampered by the low number of SSCs in the testis as well as the lack of an ideal stable long-term culture system to propagate porcine SSCs perpetually. Results In the present study, by lentiviral transduction of plasmids expressing the simian virus 40 (SV40) large T antigen into porcine primary SSCs, we developed two immortalized cell lines with porcine SSC attributes. The established cell lines, with the expression of porcine SSC and germ cell markers UCHL1, PLZF, THY1, VASA and DAZL, could respond to retinoic acid (RA), and could colonize the recipient mouse testis without tumor formation after transplantation. The cell lines displayed infinite proliferation potential, and have now been cultured for more than 7 months and passaged for over 35 times without morphological abnormalities. Conclusions We have for the first time established porcine SSC lines that could provide abundant cell sources for mechanistic studies on porcine SSC self-renewal and differentiation, thereby facilitating development of an optimal long-term culture system for porcine primary SSCs and their application to animal husbandry and medicine.

When subjected to stress, terminally differentiated neurons are susceptible to reactivate the cell cycle and become hyperploid. This process is well documented in Alzheimer’s disease (AD), where it may participate in the etiology of the disease. However, despite its potential importance, the effects of neuronal hyperploidy (NH) on brain function and its relationship with AD remains obscure. An important step forward in our understanding of the pathological effect of NH has been the development of transgenic mice with neuronal expression of oncogenes as model systems of AD. The analysis of these mice has demonstrated that forced cell cycle reentry in neurons results in most hallmarks of AD, including neurofibrillary tangles, Aβ peptide deposits, gliosis, cognitive loss, and neuronal death. Nevertheless, in contrast to the pathological situation, where a relatively small proportion of neurons become hyperploid, neuronal cell cycle reentry in these mice is generalized. We have recently developed an in vitro system in which cell cycle is induced in a reduced proportion of differentiated neurons, mimicking the in vivo situation. This manipulation reveals that NH correlates with synaptic dysfunction and morphological changes in the affected neurons, and that membrane depolarization facilitates the survival of hyperploid neurons. This suggests that the integration of synaptically silent, hyperploid neurons in electrically active neural networks allows their survival while perturbing the normal functioning of the network itself, a hypothesis that we have tested in silico. In this perspective, we will discuss on these aspects trying to convince the reader that NH represents a relevant process in AD.

Background: Spermatogonial stem cells (SSCs) are capable of both self-renewal and differentiation to mature functional spermatozoa, being the only adult stem cells in the males that can transmit genetic information to the next generation. Porcine SSCs hold great value in transgenic pig production and in establishment of porcine models for regenerative medicine. However, studies and applications of porcine SSCs have been greatly hampered by the low number of SSCs in the testis as well as the lack of an ideal stable long-term culture system to propagate porcine SSCs perpetually. Results: In the present study, by lentiviral transduction of plasmids expressing the simian virus 40 (SV40) large T antigen into porcine primary SSCs, we developed two immortalized cell lines with porcine SSC attributes. The established cell lines, with the expression of porcine SSC and germ cell markers UCHL1, PLZF, THY1, VASA and DAZL, could respond to retinoic acid (RA), and could colonize the recipient mouse testis without tumor formation after transplantation. The cell lines displayed infinite proliferation potential, and have now been cultured for more than 7 months and passaged for over 35 times without morphological abnormalities. Conclusions: We have for the first time established porcine SSC lines that could provide abundant cell sources for mechanistic studies on porcine SSC self-renewal and differentiation, thereby facilitating development of an optimal long-term culture system for porcine primary SSCs and their application to animal husbandry and medicine. [ABSTRACT FROM AUTHOR]

Abstract Background Simian Virus 40 (SV40) Large Tumor Antigen (LT) is an essential enzyme that plays a vital role in viral DNA replication in mammalian cells. As a replicative helicase and initiator, LT assembles as a double-hexamer at the SV40 origin to initiate genomic replication. In this process, LT converts the chemical energy from ATP binding and hydrolysis into the mechanical work required for unwinding replication forks. It has been demonstrated that even though LT primarily utilizes ATP to unwind DNA, other NTPs can also support low DNA helicase activity. Despite previous studies on specific LT residues involved in ATP hydrolysis, no systematic study has been done to elucidate the residues participating in the selective usage of different nucleotides by LT. In this study, we performed a systematic mutational analysis around the nucleotide pocket and identified residues regulating the specificity for ATP, TTP and UTP in LT DNA unwinding. Methods We performed site-directed mutagenesis to generate 16 LT nucleotide pocket mutants and characterized each mutant’s ability to unwind double-stranded DNA, oligomerize, and bind different nucleotides using helicase assays, size-exclusion chromatography, and isothermal titration calorimetry, respectively. Results We identified four residues in the nucleotide pocket of LT, cS430, tK419, cW393 and cL557 that selectively displayed more profound impact on using certain nucleotides for LT DNA helicase activity. Conclusion Little is known regarding the mechanisms of nucleotide specificity in SV40 LT DNA unwinding despite the abundance of information available for understanding LT nucleotide hydrolysis. The systematic residue analysis performed in this report provides significant insight into the selective usage of different nucleotides in LT helicase activity, increasing our understanding of how LT may structurally prefer different energy sources for its various targeted cellular activities.

Astrocytes, the most abundant cells in the central nervous system (CNS), are essential for neuronal development, network formation, and overall CNS homeostasis. Primary astrocyte culture has been successfully used as a tool to study astrocyte biology in vitro. In the present protocol, a modified immunopanning method was utilized to obtain and purify primary astrocytes from mouse cortex and spinal cord in a relatively quick and inexpensive way. Purified primary astrocytes were then immortalized through infection of lentivirus expressing the SV40 large T antigens. In addition, we provide protocols to determine the expression levels of astrocyte-specific markers and to perform functional studies measuring the ATP-induced calcium flux in the immortalized astrocytes. Following the described protocols assures that the immortalized astrocytes that one prepares mimic the cell biology of primary astrocytes in culture. Thus, the purification and immortalization protocols for primary astrocytes presented in here provide two models for the studies of astrocyte biology and may be useful for the immortalization of other types of primary cells. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Primary astrocyte purification by a modified immunopanning method Support Protocol: Serum-free primary astrocyte culture Basic Protocol 2: Primary astrocyte immortalization Basic Protocol 3: Calcium transient detection in astrocytes., (© 2023 Wiley Periodicals LLC.)

Dermal papilla (DP) plays important roles in hair follicle regeneration. Long-term culture of mouse DP cells can provide enough cells for research and application of DP cells. We optimized the culture strategy for DP cells from three dimensions: stepwise dissection, collagen I coating, and optimized culture medium. Based on the optimized culture strategy, we immortalized primary DP cells with SV40 large T antigen, and established several immortalized DP cell strains. By comparing molecular expression and morphologic characteristics with primary DP cells, we found one cell strain named iDP6 was similar with primary DP cells. Further identifications illustrate that iDP6 expresses FGF7 and α-SMA, and has activity of alkaline phosphatase. During the process of characterization of immortalized DP cell strains, we also found that cells in DP were heterogeneous. We successfully optimized culture strategy for DP cells, and established an immortalized DP cell strain suitable for research and application of DP cells.

The domestic pig is an important agricultural animal, and thus, infectious diseases that affect pigs can cause severe economic losses in the global swine industry. Various porcine pathogens target macrophages, which are classical innate immune cells. Although macrophages basically protect the host from pathogens, they also seem to contribute to infectious processes. Therefore, cultured macrophages can be used to develop in vitro models for studying not only genes associated with porcine innate immunity but also the infectious processes of porcine pathogens. However, the availability of porcine macrophage cell lines is limited. In this study, we describe a novel immortalized porcine kidney-derived macrophage (IPKM) cell line, which was generated by transferring the SV40 large T antigen (SV40LT) and porcine telomerase reverse transcriptase (pTERT) genes into primary porcine kidney-derived macrophages using lentiviral vectors. The IPKM displayed a typical macrophage morphology and was routinely passaged (doubling time: about 4 days). These cells were immunostained for macrophage markers. In addition, they exhibited substantial phagocytosis of polystyrene microbeads and released inflammatory cytokines upon lipopolysaccharide (LPS) stimulation. Furthermore, the maturation and secretion of interleukin-1β were observed after nigericin-induced inflammasome activation in LPS-primed IPKM. These findings suggest that IPKM exhibit the typical inflammatory characteristics of macrophages. By transferring the SV40LT and pTERT genes using lentiviral vectors, we also successfully immortalized macrophages derived from the peripheral blood of a low-density lipoprotein receptor-deficient pig. These results suggest that the co-expression of SV40LT and pTERT is an effective way of immortalizing porcine macrophages.

Background/Aims: Liver is a vital organ and retains its regeneration capability throughout adulthood, which requires contributions from different cell populations, including liver precursors and intrahepatic stem cells. To overcome the mortality of hepatic progenitors (iHPs) in vitro, we aim to establish reversibly immortalized hepatic progenitor cells from mouse embryonic liver. Methods and Results: Using retroviral system to stably express SV40 T antigen flanked with Cre/LoxP sites, we establish a repertoire of iHP clones with varied differentiation potential. The iHP cells maintain long-term proliferative activity and express varied levels of progenitor markers (Pou5f1/Oct4 and Dlk) and hepatocyte markers (AFP, Alb and ApoB). Five representative iHP clones express hepatic/pancreatic transcription factors HNF3α/Foxa1, HNF3β/Foxa2, and HNF4α/MODY1. Dexamethasone is shown to promote the expression of hepatocyte markers AFP and TAT, along with ICG-uptake and glycogen storage functions in the iHP clones. Cre-mediated removal of SV40 T antigen reverses the proliferative activity of iHP cells. When iHP cells are subcutaneously implanted in athymic nude mice, no tumor formation is observed for up to 8 weeks. Conclusions: We demonstrate that the established iHP cells are stable, reversible, and non-tumorigenic hepatic progenitor-like cells, which should be valuable for studying liver organogenesis, metabolic regulations, and hepatic lineage-specific differentiation.

Human skin is located at the outermost part of the body, and various cosmetics and chemicals that may come in contact with human skin need to be evaluated for their potential to cause irritation. Until recently, the Draize test was considered the standard method for skin irritation; however, this technique has disadvantages such as the need to sacrifice many rabbits and subjective scoring. Thus, to contribute to the development of an animal-free alternative skin irritation test, we investigated the cytotoxicity and inflammatory response to standard skin irritants in SV40 large T antigen-transformed human epidermal keratinocyte 2 cells (SV-HEK2 cells). In this study, we established an SV-HEK2 cell line immortalized by SV40 large T antigen (SV40 T) and characterized the inherent morphological and cytological properties. We next used 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) or neutral red uptake (NRU) assays of cell viability to investigate the optimal experimental conditions for determining SV-HEK2 cell viability after exposure to sodium dodecyl sulfate at 6.25 × 10 − 3 % to 1 × 10 − 1 % as a standard skin irritant. We then examined the viability of SV-HEK2 cells in response to five skin irritants (benzalkonium chloride, isopropanol, sodium dodecyl sulfate, Triton X-100 and Tween20) at 5 × 10 − 3 % to 1 × 10 − 1 % by MTT or NRU assay. Finally, we estimated the level of cytokines secretion in response to stimulation by skin irritants in SV-HEK2 cells. The results revealed that SV-HEK2 cells responded well to skin irritants in a concentration-dependent manner and that there was good correlation between irritant concentration and cytotoxicity (or cytokine secretion) when cells were exposed to skin irritants for 10 min at room temperature (RT) using an MTT assay. Overall, these findings suggest that SV-HEK2 cells could be a good alternative in vitro model for skin irritation tests. [ABSTRACT FROM AUTHOR]

Aim: S16, a monoclonal antibody against the hemagglutinin-esterase fusion (HEF) glycoprotein of influenza C virus, reacts with SV40 large T antigen (LT) and a host cellular component(s). The aim is to determine the location of S16 linear epitope on LT and the amino acid sequence of S16 epitope. Materials & methods: BHK-21 cells expressing wild-type and mutant LTs, HEFs or GFPs, each of which was tagged with a FLAG epitope, were analyzed by immunoblotting using S16. Results & conclusions: An amino acid sequence 98-FNEENL-103 on LT forms a linear epitope recognized by S16. The sequence of S16 epitope was defined as F[NAT]EE[NYA]L, excluding FAEEAL and FTEEAL. This finding will be of help in identifying a host cellular component(s) crossreactive with S16. [ABSTRACT FROM AUTHOR]

Cellular eukaryotic replication initiation helicases are first loaded as head-to-head double hexamers on double-stranded (ds) DNA origins and then initiate S-phase DNA melting during licensed (once per cell cycle) replication. Merkel cell polyomavirus (MCV) large T (LT) helicase oncoprotein similarly binds and melts its own 98-bp origin but replicates multiple times in a single cell cycle. To examine the actions of this unlicensed viral helicase, we quantitated multimerization of MCV LT molecules as they assembled on MCV DNA origins using real-time single-molecule microscopy. MCV LT formed highly stable double hexamers having 17-fold longer mean lifetime (τ, >1,500 s) on DNA than single hexamers. Unexpectedly, partial MCV LT assembly without double-hexamer formation was sufficient to melt origin dsDNA as measured by RAD51, RPA70, or S1 nuclease cobinding. DNA melting also occurred with truncated MCV LT proteins lacking the helicase domain, but was lost from a protein without the multimerization domain that could bind only as a monomer to DNA. SV40 polyomavirus LT also multimerized to the MCV origin without forming a functional hexamer but still melted origin DNA. MCV origin melting did not require ATP hydrolysis and occurred for both MCV and SV40 LT proteins using the nonhydrolyzable ATP analog, adenylyl-imidodiphosphate (AMP-PNP). LT double hexamers formed in AMP-PNP, and melted DNA, consistent with direct LT hexamer assembly around single-stranded (ss) DNA without the energy-dependent dsDNA-to-ssDNA melting and remodeling steps used by cellular helicases. These results indicate that LT multimerization rather than helicase activity is required for origin DNA melting during unlicensed virus replication.

The simian virus 40 large T antigen (SVLT) induces replication of plasmids bearing the SV40 origin of replication (SV40 ori) within mammalian cells. The internal ribosomal entry site (IRES) is an element that allows for the cotranslation of proteins from one polycistronic mRNA. Through the combination of these elements, IRES-dependent coexpression of a protein of interest and the SVLT, either constitutive or regulated, on plasmids bearing the SV40 ori generates a positive feedback loop, resulting in enhanced expression. A vector linking red fluorescent protein (RFP) to the IRES-SVLT element enhances fluorescence ∼10-fold over that demonstrated from a vector lacking this element. In transfection-resistant CV-1 cells, the RFP-IRES-SVLT vector substantially increases the number of cells expressing detectable levels of RFP. Furthermore, inclusion of the IRES-SVLT/SV40 ori elements in standard luciferase-based reporter gene constructs and associated effectors results in marked increases in luminescent output and sensitivity, using the β-catenin/TCF pathway and the mammalian two-hybrid assay as models. Ultimately, vector systems combining these well-established elements (IRES-SVLT/SV40 ori) will increase the utility of transient transfection for the production of recombinant proteins, the use of transfection-resistant cell lines, and the effectiveness of luciferase-based high-throughput screening assays.

Introduction Primary cultures of human corneal epithelial (HCE) cells usually cease to grow after four or five passages. This result in a small cell yield for experiments such as the eye irritancy test represents a serious problem for human and animal corneal epithelial research. In the present study, we established an HCE cell line immortalized by simian virus 40 (SV40), a polyomavirus, and characterized the inherent morphologic and cytologic cell properties. Methods Primary cultured HCE cells were infected with a SV40 large T antigen (SV40 T)-expressing retrovirus, and were selected using G418 solution, an aminoglycoside antibiotic. To ensure that the immortalized cell lines express SV40 T and cytokeratin-3, a corneal epithelial-specific marker, we conducted reverse-transcription (RT)-PCR and Western blot analysis. Results These cell lines continued to grow for more than 50 generations, exhibiting a cobble stone-like appearance similar to normal HCE cells and an increased proliferation rate compared to primary cultured HCE cells. RT-PCR results showed that the immortalized cell lines expressed SV40 T while the primary cultured cells did not. In the Western blot assay, protein levels of phosphorylated (Ser15) p53 protein were significantly decreased in the immortalized cell lines while the expression of total p53 protein was constant. In addition, expression of p21 cip1 , a cell cycle protein, was down-regulated in the immortalized cells. Moreover, a cornea epithelium-specific marker, cytokeratin-3 (CK-3), was expressed at equal levels in the immortalized cells and primary HCE cells. Discussion Taken together, these results indicate that immortalized HCE cell lines were successfully established using the SV40-retroviral vector. These cells may be an excellent model for detecting the adverse effects of standard toxic materials and could replace the traditional eye irritation test as an animal-free alternative method. [ABSTRACT FROM AUTHOR]

Background: The MexTAg transgenic mouse model of mesothelioma replicates many aspects of human mesothelioma, including induction by asbestos, pathogenicity and response to cytotoxic chemotherapy, despite high levels of the SV40 large T Antigen (TAg) in the mesothelial compartment. This model enables analysis of the molecular events associated with asbestos induced mesothelioma and is utilised here to investigate the molecular dynamics of tumours induced in these mice, using gene expression patterns as a read out.Methods: Gene expression of MexTAg mesothelioma cell lines bearing a high or low number of copies of the TAg transgene were compared to wild type mouse mesotheliomas and normal mouse mesothelial cells using Affymetrix microarray. These data were then compared to a similar published human microarray study using the same platform.Results: The main expression differences between transgenic mouse and wild type mouse mesotheliomas occurred for genes involved in cell cycle regulation and DNA replication, as would be expected from overexpression of the TAg oncogene. Quantitative PCR confirmed that E2F and E2F regulated genes were significantly more upregulated in MexTAg mesotheliomas and MexTAg mesothelial cells compared to wild type mesotheliomas. Like human mesothelioma, both MexTAg and wild type mesotheliomas had more genes underexpressed than overexpressed compared to normal mouse mesothelial cells. Most notably, the cdkn2 locus was deleted in the wild type mouse mesotheliomas, consistent with 80 % human mesotheliomas, however, this region was not deleted in MexTAg mesotheliomas. Regardless of the presence of TAg, all mouse mesotheliomas had a highly concordant set of deregulated genes compared to normal mesothelial cells that overlapped with the deregulated genes between human mesotheliomas and mesothelial cells.Conclusions: This investigation demonstrates that the MexTAg mesotheliomas are comparable with wild type mouse mesotheliomas in their representation of human mesothelioma at the molecular level, with some key gene expression differences that are attributable to the TAg transgene expression. Of particular note, MexTAg mesothelioma development was not dependent on cdkn2 deletion. [ABSTRACT FROM AUTHOR]

Aim Polyomavirus BK nephropathy ( BKVN) is an important complication in kidney transplantation. After immunosuppressive agents are reduced, some patients experience a temporal increase in serum creatinine ( sCr) before viral clearance. The histological characteristics of re-biopsies were therefore investigated to evaluate the time course of remission. Methods sCr was measured and urinary cytology evaluated periodically in 14 patients with biopsy-proven BKVN. Remission of BKVN was defined as re-biopsies negative for SV40 large T antigen ( SV40- TAg) or for decoy cells on at least three consecutive cytology tests. Early changes in sCr were correlated with re-biopsy findings. Results Mean sCr was 1.6 ± 0.6 mg/dL at diagnosis, increasing during the first 2 months to 2.6 ± 2.0 mg/dL, and decreasing thereafter, to 2.3 ± 1.2 mg/dL at 3-4 months. Two patients who experienced further increases in sCr at 3 months showed early graft loss, while the others showed clinical or histological remission. Nineteen re-biopsies were obtained from eight patients over 4 months. Banff i-scores were higher in re-biopsies obtained during the first 2 months than the index biopsies and re-biopsies at 2-4 months ( P = 0.02). SV40- TAg positivity was common in re-biopsies during the first 2 months (10/11 biopsies), but rarer at 2-4 months (2/8 biopsies, P = 0.001). Conclusions Temporal graft dysfunction and increased inflammation, called immune reconstitution, were observed at 2 months. Later sCr reversal is associated with remission. [ABSTRACT FROM AUTHOR]

Objectives: Due to the rarity of human embryonic samples and limited proliferating capability of primary human dental mesenchymal cells, it is valuable to create an immortalized human dental mesenchymal cell line for studying dental mesenchymal cell differentiation and signalling pathways during detinogenesis in humans. Methods: In this study, dental mesenchymal cells from human molar tooth germs at 19- week gestation were isolated and immortalized with pSV40. Single cell colonies were then selected by 96-well plate dilution. The immortalized cell line was characterized using immmunofluorescent microscopy, RT-PCR and Western blot for the expression of SV40 large T antigen and five genes specific for the mesenchymal stage during tooth development. The differentiation and mineralization activities of the immortalized and primary cells were compared using adipogenic and calcifying induction. Results: The immortalized dental mesenchymal cell line displayed a higher proliferation rate, expressed several tooth-specific markers including Msx1, Pax9, Lhx6, Barx1, and Runx2, and maintained the ability to differentiate and form mineralized nodules. Conclusions: Our results demonstrated that the immortalized human mesenchymal cell line retained the characteristics similar to primary human dental mesenchymal cells and can be used for studying the mechanisms of human dental mesenchymal cell differentiation and signalling pathways involved in human odontogenesis. [ABSTRACT FROM AUTHOR]

Background/Aims: Liver is a vital organ and retains its regeneration capability throughout adulthood, which requires contributions from different cell populations, including liver precursors and intrahepatic stem cells. To overcome the mortality of hepatic progenitors (iHPs) in vitro, we aim to establish reversibly immortalized hepatic progenitor cells from mouse embryonic liver. Methods and Results: Using retroviral system to stably express SV40 T antigen flanked with Cre/LoxP sites, we establish a repertoire of iHP clones with varied differentiation potential. The iHP cells maintain long-term proliferative activity and express varied levels of progenitor markers (Pou5f1/Oct4 and Dlk) and hepatocyte markers (AFP, Alb and ApoB). Five representative iHP clones express hepatic/pancreatic transcription factors HNF3α/Foxa1, HNF3β/Foxa2, and HNF4α/MODY1. Dexamethasone is shown to promote the expression of hepatocyte markers AFP and TAT, along with ICG-uptake and glycogen storage functions in the iHP clones. Cre-mediated removal of SV40 T antigen reverses the proliferative activity of iHP cells. When iHP cells are subcutaneously implanted in athymic nude mice, no tumor formation is observed for up to 8 weeks. Conclusions: We demonstrate that the established iHP cells are stable, reversible, and non-tumorigenic hepatic progenitor-like cells, which should be valuable for studying liver organogenesis, metabolic regulations, and hepatic lineage-specific differentiation. © 2014 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

The murine preosteoblastic cell line, MC3T3-E1, is widely used to study bone formation and differentiation in vitro. However, this cell line is unstable in culture. The current study was designed to establish a stable osteoblastic cell line. A mammalian expression vector carrying the SV 40 large T antigen was introduced into a primary culture of cells isolated from the calvaria of newborn mice. Among isolated cell lines, the MN16 cell line was selected for further characterization. The MN16 cell line was cultured for 28 days, and compared with the MC3T3-E1 cell line with or without induction. The expression of bone-related genes was examined using the real-time RT-PCR technique. Alizarin red and von Kossa staining were used to detect mineralization of nodules in the cultures. The cell line showed the characteristics of osteoblastic cells in term of gene expression patterns of various molecular markers and calcium deposition in the cell layer after induction. Furthermore, the MN16 cells showed strong adhesion to the basic domain of collagen, a result that is specific for bone-derived cells. The MN16 cell line was found to be stably differentiated into bone formation cells in vitro and should be useful for studying bone biology. [ABSTRACT FROM AUTHOR]

Human Hertwig's epithelial root sheath/epithelial rests of Malassez (HERS/ERM) cells are epithelial remnants of teeth residing in the periodontium. Although the functional roles of HERS/ERM cells have yet to be elucidated, they are a unique epithelial cell population in adult teeth and are reported to have stem cell characteristics. Therefore, HERS/ERM cells might play a role as an epithelial component for the repair or regeneration of dental hard tissues; however, they are very rare population in periodontium and the primary isolation of them is considered to be difficult. To overcome these problems, we immortalized primary HERS/ERM cells isolated from human periodontium using SV40 large T antigen (SV40 LT) and performed a characterization of the immortalized cell line. Primary HERS/ERM cells could not be maintained for more than 6 passages; however, immortalized HERS/ERM cells were maintained for more than 20 passages. There were no differences in the morphological and immunophenotypic characteristics of HERS/ERM cells and immortalized HERS/ERM cells. The expression of epithelial stem cell and embryonic stem cell markers was maintained in immortalized HERS/ERM cells. Moreover, immortalized HERS/ERM cells could acquire mesenchymal phenotypes through the epithelial-mesenchymal transition via TGF-β1. In conclusion, we established an immortalized human HERS/ERM cell line with SV40 LT and expect this cell line to contribute to the understanding of the functional roles of HERS/ERM cells and the tissue engineering of teeth. [ABSTRACT FROM AUTHOR]

Highlights: [•] Transient expression of antibodies up to 400mg/L. [•] Experimental design techniques lead to an optimal process. [•] A combination of SV40 large T antigen, p21 and p27 increases expression levels. [•] Fully scalable from 0.1 up to 1200ml. [ABSTRACT FROM AUTHOR]

Abstract: Methylmercury (MeHg) is a persistent environmental contaminant with potent neurotoxic action for which the underlying molecular mechanisms remain to be conclusively delineated. Our objectives herein were twofold: first, to corroborate our previous findings of an increased sensitivity of spontaneously-immortalized oxoguanine glycosylase 1-null (Ogg1−/− ) murine embryonic fibroblasts (MEFs) to MeHg through generation of Simian virus 40 (SV40) large T antigen-immortalized wild-type and Ogg1−/− MEFs; and second, to determine whether MeHg toxicity is proliferation-dependent. As with the spontaneously-immortalized cells used previously, the SV40 large T antigen-immortalized cells exhibited similar tendencies to undergo MeHg-initiated cell cycle arrest, with increased sensitivity in the Ogg1−/− MEFs as measured by clonogenic survival and DNA damage. Compared to exponentially growing cells, those seeded at a higher density exhibited compromised proliferation, which proved protective against MeHg-mediated cell cycle arrest and induction of DNA double strand breaks (DSBs), measured by phosphorylation of the core histone H2A variant (H2AX) on serine 139 (γH2AX), and by its functional confirmation by micronucleus assessment. This enhanced sensitivity of Ogg1−/− MEFs to MeHg toxicity using discrete SV40 immortalization corroborates our previous studies, and suggests a novel role for OGG1 in minimizing MeHg-initiated DNA lesions that trigger replication-associated DSBs. Furthermore, proliferative capacity may determine MeHg toxicity in vivo and in utero. Accordingly, variations in cellular proliferative capacity and interindividual variability in repair activity may modulate the risk of toxicological consequences following MeHg exposure. [Copyright &y& Elsevier]

Background: The African spiny mouse ( Acomys ) is an emerging mammalian model for scar-free regeneration, and further study of Acomys could advance the field of regenerative medicine. Isolation of pluripotent stem cells from Acomys would allow for development of transgenic or chimeric animals and in vitro study of regeneration; however, the reproductive biology of Acomys is not well characterized, complicating efforts to derive embryonic stem cells. Thus, we sought to generate Acomys induced pluripotent stem cells (iPSCs) by reprogramming somatic cells back to pluripotency. Methods: To generate Acomys iPSCs, we attempted to adapt established protocols developed in Mus . We utilized a PiggyBac transposon system to genetically modify Acomys fibroblasts to overexpress the Yamanaka reprogramming factors as well as mOrange fluorescent protein under the control of a doxycycline-inducible TetON operon system. Results: Reprogramming factor overexpression caused Acomys fibroblasts to undergo apoptosis or senescence. When SV40 Large T antigen (SV40 LT) was added to the reprogramming cocktail, Acomys cells were able to dedifferentiate into pre-iPSCs. Although use of 2iL culture conditions induced formation of colonies resembling Mus PSCs, these Acomys iPS-like cells lacked pluripotency marker expression and failed to form embryoid bodies. An EOS-GiP system was unsuccessful in selecting for bona fide Acomys iPSCs; however, inclusion of Nanog in the reprogramming cocktail along with 5-azacytidine in the culture medium allowed for generation of Acomys iPSC-like cells with increased expression of several naïve pluripotency markers. Conclusions: There are significant roadblocks to reprogramming Acomys cells, necessitating future studies to determine Acomys -specific reprogramming factor and/or culture condition requirements. The requirement for SV40 LT during Acomys dedifferentiation may suggest that tumor suppressor pathways play an important role in Acomys regeneration and that Acomys may possess unreported cancer resistance., Competing Interests: No competing interests were disclosed., (Copyright: © 2022 Sandoval AGW et al.)

Transplantation is common in clinical practice where there is availability of the tissue and organ. In the case of neurodegenerative disease such as Parkinson's disease ( PD), transplantation is not possible as a result of the non-availability of tissue or organ and therefore, cell therapy is an innovation in clinical practice. However, the availability of neuronal cells for transplantation is very limited. Alternatively, immortalized neuronal progenitors could be used in treating PD. The neuronal progenitor cells can be differentiated into dopaminergic phenotype. Here in this article, the current understanding of the molecular mechanisms involved in the differentiation of dopaminergic phenotype from the neuronal progenitors immortalized with SV40 LT antigen is discussed. In addition, the methods of generating dopaminergic neurons from progenitor cells and the factors that govern their differentiation are elaborated. Recent advances in cell-therapy based transplantation in PD patients and future prospects are discussed. [ABSTRACT FROM AUTHOR]

The article presents a study which examined the changes in gene expression in medulloblastoma and tumor-associated genes in the sonic hedgehog, Wnt/beta-catenin and insulin-like growth factor (IGF) signaling pathways. Gene expression changes were examined through microarray analysis. The study concludes that the simian virus 40 large T antigen plays an important role in the development of medulloblastomas and may cooperate with IGF pathway components.

We transformed mous podocytes by ectopic expression of cyclin-dependent kinase 4 (CDK4). Compared with podocytes transformed with a thermo-sensitive SV40 large T antigen mutant tsA58U19 (tsT podocytes), podocytes transformed with CDK4 (CDK4 podocytes) exhibited significantly higher expression of nephrin mRNA. Synaptopodin mRNA expression was significantly lower in CDK4 podocytes and in tsT podocytes under growth-permissive conditions (33°C) compared with tsT podocytes under growth-restricted conditions (37°C), which suggests a role for cell cycle arrest in synaptopodin mRNA expression. Confluent CDK4 podocytes showed significantly higher mRNA expression levels for nephrin, synaptopodin, Wilms tumor I, podocalyxin, and P-cadherin compared with subconfluent cultures. We carried out experiments to clarify roles of various factors in the confluent podocyte cultures; our findings indicate that cell-cell contact promotes expression of five podocyte marker genes studied, that cellular quiescence increases synaptopodin and podocalyxin mRNA expression, and that soluble factors play a role in nephrin mRNA expression. Our findings suggest that CDK4 podocytes are useful tools to study podocyte biology. Furthermore, the role of cell-cell contact in podocyte gene expression may have relevance for podocyte function in vivo. [ABSTRACT FROM AUTHOR]

Neuronal progenitor cells (NPCs) possess high potential for use in regenerative medicine. To overcome their limited mitotic competence, various immortalization strategies have been applied that allow their prolonged maintenance and expansion in vitro. Such immortalized cells can be used for the design and discovery of new cell-based therapies for neurodegenerative diseases, such as Parkinson’s disease. We immortalized rat ventral mesencephalic NPCs by using SV40 large T antigen (SV40Tag). All cell clones displayed a two- to three–fold higher proliferation rate compared with the primary cells. In order to induce dopaminergic differentiation of generated cell clones, both glial-derived neurotrophic factor and di-butyryl cyclic adenosine monophosphate were applied. Treated cells were then characterized regarding the expression of dopaminergic lineage markers, differentiation of various cell populations, calcium imaging in the presence of kainate, and immunohistochemistry after intrastriatal transplantation. Treated cells displayed morphological maturation, and calcium imaging revealed neuronal properties in the presence of kainate. These cells also expressed low mRNA levels of the dopamine transporter and tyrosine hydroxylase (TH), although no TH-immunopositive neurons were found. Intrastriatal transplantation into the neurotoxin-lesioned rats did not induce further differentiation. As an alternative approach, we silenced SV40Tag with short interfering RNA, but this was not sufficient to trigger differentiation into dopaminergic neurons. Nevertheless, neuronal and glial cells were detected as shown by β-tubulin type III and glial fibrillary acidic protein staining, respectively. SV40Tag cells are suitable for carrying out controlled genetic modifications as shown by overexpression of enhanced green fluorescence protein after efficient non-viral transfection. [ABSTRACT FROM AUTHOR]

Evidence suggests that loss of podocytes into urine contributes to development of glomerular diseases; shed podocytes are frequently viable and proliferate in culture conditions. To determine the phenotypic characteristics of viable urinary cells derived from human subjects, we established long-term urinary cell culture from two patients with focal segmental glomerulosclerosis and two healthy volunteers, via transformation with the thermosensitive SV40 large T antigen (U19tsA58) together with human telomerase (hTERT). Characterization of arbitrarily selected two clonal cell lines from each human subject was carried out. mRNA expression for the podocyte markers synaptopodin, nestin, and CD2AP were detected in all eight clones. Podocin mRNA was absent from all eight clones. The expression of nephrin, Wilms' tumor 1 (WT1), and podocalyxin mRNA varied among the clones, which may be due to transformation and/or cloning. These results suggest that podocyte cell lines can be established consistently from human urine. The generation of podocyte cell lines from urine of patients and healthy volunteers is novel and will help to advance studies of podocyte cell biology. Further improvements in the approaches to cell transformation and/or cell culture techniques are needed to allow cultured podocytes to fully reproduce in vivo characteristics. [ABSTRACT FROM AUTHOR]

Aim To establish and characterize different types of fibroblastic cell lines derived from dental pulp tissue. Methodology Human dental pulp tissue-derived cells were transfected with SV40 large T antigen by Lipofectamine transfection method. Geneticin (G418)-resistant cells were selected and different cell lines were established by a limiting dilution method. To characterize the lineages of cells, each clone was immunofluorescently stained by anti-fibroblast, anti-vimentin, anti-collagen type I and type III antibodies. Total RNA was extracted from each clone and subjected to a differential display experiment. Results By transfecting SV40 large T antigen, nine different cell clones were obtained. All these cell clones were positively stained by anti-fibroblast, anti-vimentin, anti-collagen type I and type III antibodies. With differential display experiment, eight different genes, the expression levels of these genes were varied amongst each cell clone, were detected. After sequencing and database search, one gene was revealed to be identical to T-cell marker, Thy-1. Thy-1 expression in dental pulp tissue was confirmed by immunohistochemical staining. Conclusion Fibroblastic cell lines derived from human dental pulp tissue possessed different gene expression profiles suggesting the existence of subpopulations. [ABSTRACT FROM AUTHOR]